Photodynamic therapy (PDT) generally involves the administration of compounds that are capable of absorbing light, typically in the visible range, but also in the near ultraviolet, followed by irradiation of locations in the subject for which a toxic, inhibitory or modulatory effect is desired. PDT was initially developed using hematoporphyrin and related compounds in the treatment of tumors, as it appeared that these compounds would “home” to locations containing rapidly dividing cells. The tumor could then be irradiated with light absorbed by the hematoporphyrin and destruction of the surrounding tissue resulted (for example, see U.S. Pat. Nos. 4,932,934 and 5,283,255). PDT has since been shown to be useful for treatment of my other conditions, including ocular diseases characterized by unwanted neovascularization, such as aged-related macular degeneration (see U.S. Pat. Nos. 5,756,541 and 5,798,349), the inhibition of secondary cataract formation in the eye (U.S. Pat. No. 6,043,237), the impairment of blood-borne targets such as leukemic cells and immunoreactive cells (U.S. Pat. Nos., 5,776,966, 5,807,881 and 5,868,695) the removal of unwanted microorganisms (U.S. Pat. No. 5,360,734), the removal of atherosclerotic plaque (U.S. Pat. No. 5,834,503) as well as the prevention of transplant rejection by pre-treating the graft tissue (U.S. Pat. No. 5,882,328).
The search for effective photosensitizers requires a two-pronged approach. The optimization of photophysical properties is key to any promising drug as the compounds must absorb at long wavelengths. The development of higher wavelength photosensitizers requires a synthetic method which can generate a number of analogs with ease because in vivo biological proficiency is known to increase on going from porphyrins to chlorins to bacteriochlorins. Compounds must also display good biodistribution properties in order to be effective. The correlation between the biodistribution of photosensitizers and the structure of the drug is complex. This complexity is increased with hydrophobic molecules which must be formulated into a suitable transport system such as liposomes, emulsions or nanoparticles. The delivery systems of these drugs are crucial and have been a key obstacle in PDT. These systems are complicated in that their nature drastically affects both the rate and the amount of drug taken up by the cells.
A very large percentage of porphyrin-based photosensitizers are transported via protein binding. For example, at least 95% of hematoporphyrin (Hp) at the normal dose used for PDT (3–5 mg/kg body weight) is complexed by serum proteins (Jori, G. in Photosensitizing Compounds: their Chemistry, Biology and Clinical Use. Wiley, Chichester. Ciba Foundation Symposium 1989, 146, 79). Human serum consists of three protein fractions: lipoproteins (high density (HDL), low density (LDL) and very low density (VLDL)), globulins and albumin. The distribution of photosensitizers in the serum is strongly dependent upon their chemical structure. Hydrophilic, polar photosensitizers are bound preferentially by albumin and globulins whilst hydrophobic molecules are bound by lipoproteins (Ochsner, M. Arzneim.-Forsch./Drug Res. 1997, 47(II), 1185–1194).
Albumin and globulins are known to possess a distinct number of binding sites (Kessel, D. Cancer Lett. 1986, 33, 183). The binding of photosensitizer molecules to albumin and globulin is governed by a chemical equilibrium between the bound and unbound photosensitizer (Supra). In contrast, the binding of hydrophobic dyes to lipoproteins reflects a partition of the photosensitizer between the lipid and the aqueous phase and therefore many photosensitizer molecules can bind to each lipoprotein. The relative binding of tetrapyrroles to lipoproteins has been shown to increase with decreasing polarity (Bonnett, R. SPIE 1993, 2078, 74). The partitioning of hydrophobic photosensitizers is significant as these dyes tend to aggregate in aqueous systems. The extent of aggregation is dependent upon the polarity of the substituents on the porphyrin skeleton (Redmond, R. W.; Land, E. J.; Truscott, T. G. in Advances in Experimental Medicine and Biology. Volume 193. Kessel, D. Ed.; Plenum, N.Y., 1985, 293). Only monomeric nonaggregated molecules are photoactive and therefore any aggregation will decrease the observed cytotoxicity of the drug (Ibid, p. 301).
Hydrophobic photosensitizers must, therefore, be properly formulated in order to counteract their natural tendency to aggregate in aqueous systems. An advantage of hydrophobic drugs is their preferential binding to lipoproteins as tumor cells express a much larger number of receptors for low density lipoproteins (LDL) than do most normal cells (Spikes, J. D. in Light in Biology and Medicine Vol. 1. Douglas, R. A.; Moan, J.; Dall'Acqua F. Eds.; Plenum, N.Y., 1988, p. 105). These receptors specifically recognize LDL and promote their internalization by cells via the formation of coated pits. Photosensitizers that bind to LDL are endocytosed by the neoplastic cells along with the lipoprotein (Fisher, A. M. R.; Murphree, A. L.; Gomer, C. J. Lasers in Surgery and Medicine 1993, 17, 2). Once inside the cell, the photosensitizer is released into the cytoplasm and binds to apolar endocellular matrices such as mitochondria, lysosomes and plasma membranes. A photosensitizer will be most effective if it displays an affinity for tumor cells versus normal cells because low cytotoxicity of such a drug can be overcome by increasing the dose. In the early 1980s, ortho-, meta- and para-isomers of meso-tetra(hydroxyphenyl)porphyrin were investigated for use as photosensitizers (Berenbaum, M. C.; Akande, S. L.; Bonnett, R.; Kaur, H.; Ioannou, S.; White, R. D.; Winfield, U.-J. Br. J. Cancer 1986, 54, 717). In order to increase the absorption in the red region, the analogous chlorins and the meta-hydroxy substituted bacteriochlorin were synthesized (Bonnett, R.; Berenbaum, M. in Photosensitizing Compounds: their Chemistry, Biology and Clinical Use. Wiley, Chichester. Ciba Foundation Symposium 1989, 146, 40–59). In vivo testing showed that both phototoxicity (reflected by the decreased dose) and tissue penetration (reflected by the increased depth of necrosis) increased as did the level of reduction of the porphyrin (Bonnett, R. Proc. SPIE 1995, 2371, 31). Tetra(m-hydroxyphenyl)chlorin was chosen as the most suitable for clinical trials and was found to be 25–30 times more effective than HpD in destroying tumors as observed by in vivo bioassays with LD50=3 mg/kg (Bonnett, R.; Berenbaum, M. Br. J. Cancer 1991, 64, 1116). Tetra(m-hydroxyphenyl)chlorin showed 90% tumor necrosis with only 10% recurrence, but side effects such as extended skin sensitivity, severe chest pains and loss of appetite were also observed.
The β,β′-dihydroxylation of meso-tetraphenylporphyrins and mesotetraphenyl chlorins via osmium tetroxide mediated oxidation has been previously described and patented (Bruckner, C.; Dolphin, D. Tetrahedron Lett. 1995, 36, 9425; and Bruckner, C.; Dolphin, D. Tetrahedron Lett. 1995, 36, 3295) and U.S. Pat. No. 5,648,485 issued Nov. 3, 1998, which is hereby incorporated by reference as if fully set forth.